1. Field of the Invention
The present invention concerns an improved method for immunizing against hepatitis B virus (HBV) by including in a hepatitis B virus vaccine one or more antigens of non-permitted variant sequences within residues S(139-147) of the hepatitis B virus surface antigen.
2. Background Information
There are approximately 600,000 persistent carriers of hepatitis B virus (HBV) in the United States; the estimated total number of carriers in the world is 300 million. A considerable portion of HBV carriers have chronic liver disease. The involvement of HBV in liver cancer has been demonstrated (W. Szmuness, Prog. Med. Virol. 24, 40 (1978) and R. P. Beasley, L.-Y. Hwang, C. -C. Ling, C.-S. Chien, Lancet, Nov., 21, 1129 (1981)).
HBV infections thus represent a major public health problem worldwide. Already available vaccines (S. Krugman, in Viral Hepatitis: Laboratory and Clinical Science, F. Dienhardt, J. Dienhardt, Eds. Marcel Dekker, Inc., New York-Basel, 1983, pp. 257-263) produced from the serum of HBV carriers, because of limited resources and production costs involved, do not provide the appropriate means to control and eradicate the disease worldwide. There is hope, however, that this may be accomplished by vaccines based on recombinant DNA technology and/or synthetic peptides.
The biology, structure and immunochemistry of HBV and the genetic organization of its DNA genome have been reviewed (B. S. Blumberg, Science, 197, 17, (1977)). The cloning and sequencing of the genome of several hepatitis virus (HBV) isolates led to the elucidation of the genetic structure of the viral DNA (P. Tiollais, P. Charnay, G. N. Vyas, Science, 213, 406, (1981)).
The immunologic markers of HBV infection include the surface antigen (HBsAg), the core antigen (HBcAg), the "e" antigen (HBeAg) and their respective antibodies. Antibodies against HBsAg are protective against HBV infection.
Several antigenic subtypes of HBV and of subviral approximately 22 nm diameter particles (hepatitis B surface antigen; HBsAg) have been recognized (G. Le Bouvier, A. Williams, Am. J. Med. Sci., 270,.165 (1975)). All of these subtypes (for example, ayw, adyw, adw2, adw and adr) share common (group-specific) envelope epitopes, the immune response against which appears sufficient for protection against infection by any of the virus subtypes (W. Szmuness, C. E. Stevens, E. J. Hartley, E. A. Zang, H. J. Alter, P. E. Taylor, A. DeVera, G. T. S. Chen, A. Kellner et al, N. Enql. J. Med., 307, 1481, (1982)).
Since dominant B-cell epitopes on the S-protein of the hepatitis B virus surface antigen (HBsAg) are discontinuous, it has proved difficult to mimic them by linear synthetic peptides. However, some cyclic peptides derived from segments of the hepatitis B virus surface antigen sequence were shown to bind anti-HBs.
A cyclic peptide corresponding to the sequence S(139-147), derived from the sequence of the S-protein subtype adw, with a disulfide bond between residues (139-147) was reported to bind anti-HBs with an affinity similar to that determined for the reaction between native HBsAg and anti-HBs (S. E. Brown, C. R. Howard, A. J. Zuckerman and M. W. Steward, 1984, "Determination of the Affinity of Antibodies to Hepatitis B Surface Antigen in Human Sera", J. Immunol. Methods, 72:41-48; S. E. Brown, A. J. Zuckerman, C. R. Howard and M. W. Stewart, 1984, "Affinity of Antibody Responses in Man to Hepatitis B Vaccine Determined With Synthetic Peptides", Lancet, 2:184-187. The same peptide after polymerization with glutaraldehyde, elicited antibodies reactive with native HBsAg (C. R. Howard, J. Allan, S. -H. Chen, S. E. Brown and M. H. Steward, 1986, "Progress Toward a Synthetic Hepatitis B Vaccine", pp. 133-136. In H. Peeters (Ed), Protides of the Biological Fluids: Proceedings of the Thirty-Fourth Colloquium, Pergamon Press, Oxford, England). These results indicate that the sequence S(139-147) is a portion of a dominant discontinuous B-cell epitope of the S-protein.
The peptide S(139-147) in both linear and cyclic forms elicited the proliferation of T-helper (T.sub.h) lymphocytes from mice and humans immunized with HBsAg (Neurath, A. R. and Y. Thanavala, 1990 supra. These results indicate that the S(139-147) segment of S-protein is part of an immunologically important region recognized by both B and T.sub.h cells.
Since the S(139-147) segment of the S-protein sequence is important for eliciting HBsAg-specific B and T.sub.h -cell responses, amino acid replacements within this sequence may profoundly affect the recognition of the S-protein by both B- and T.sub.h -cells and the specificity of immune responses to the S-protein. Among well-defined serological-subtypes of HBsAg there is a single amino acid substitution (serine threonine) at residue 143. All other amino acid residues within this sequence are completely conserved among the distinct HBV subtypes.
Evidence for the existence of genetic variants of HBV with envelope protein epitopes distinct from those present on already defined HBV subtypes has been reported recently M. E. Lai, P. Farci, A. Figus, A. Balestrieri, M. Arnone and G. N. Vyas, 1989, "Hepatitis B Virus DNA in the Serum of Sardinian Blood Donors Negative for the Hepatitis B Surface Antigen", Blood, 73:17-19. Direct evidence for the emergence of such variants under immunological pressure in vivo comes from recent studies of McMahon et al (G. McMahon, L. A. McCarthy, D. Dottavio and L. Ostberg, not yet published "Surface Antigen and Polymerase Gene Variation in Hepatitis B Virus Isolates from a Monoclonal Antibody Treated Liver Transplant Patient", B. Hollinger (Ed), Proceedings of the 1990 International Symposium on Viral Hepatitis and Liver Disease, Wiley, N.Y., U.S.A.).
Amino acid replacements within the S-protein sequence may lead to a loss of subtype specific determinants d/y or w/r (H. Okamoto, S. Omi, Y. Wang, Y. Itoh, F. Tsuda, T. Tanaka, Y. Akahane, Y. Miyakawa and M. Mayumi, 1989, "The Loss of Subtypic Determinants in Alleles, d/y or w/r, on Hepatitis B Surface Antigen", Mol. Immunol., 26:197-205). However, these newly discerned HBV subtypes, which are nonreactive with subtype specific reagents developed earlier, still contain the group specific "a" determinants considered essential for eliciting protective immunity (Neurath, 1989, "Chemical Synthesis of Hepatitis B Vaccines", p. 210-242, In A. J. Zuckerman (Ed), Recent Developments in Prophylactic Immunization, Kluwer Academic Publishers, Dordrecht, The Netherlands; Neurath and Thanavala, 1990, "Hepadnaviruses", In M. H. V. Van Regenmortel and A. R. Neurath (Eds), Immunochemistry of Viruses II, Elsevier Science Publishers, Amsterdam, The Netherlands, in press). However, HBV variants may have altered or insufficiently cross-reactive a determinants recognizable by antibodies and T cells elicited as a result of immunization with defined subtypes of HBV. Such variants may possibly cause infections not preventable by current hepatitis B vaccines. For this reason, it is important to define amino acid replacements within dominant group-specific B and T cell epitopes which would lead to the generation of escape mutants.
Antigenic variation with respect to foot-and-mouth disease virus is discussed in Immunochemistry of Viruses. The Basis for Serodiagnosis and Vaccines, Ed. M. H. V. Van Regenmortel and A. R. Neurath, "Antigenic Structure of Foot-And-Mouth Disease Virus", F. Brown, 274-276, (1985).
DEFINITIONS Amino Acid 3-letter code 1-1etter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutainine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V